title: Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis.
abstract: Tuberculosis is a major cause of mortality and morbidity due to infectious disease. However, current clinical diagnostic methodologies such as PCR, sputum culture, or smear microscopy are not ideal. Antibody-based assays are a suitable alternative but require specific antibodies against a suitable biomarker. Mycolic acid, which has been found in patient sputum samples and comprises a large portion of the mycobacterial cell wall, is an ideal target. However, generating anti-lipid antibodies using traditional hybridoma methodologies is challenging and has limited the exploitation of this lipid as a diagnostic marker. We describe here the isolation and characterization of four anti-mycolic acid antibodies from a nonimmune antibody phage display library that can detect mycolic acids down to a limit of 4.5ng. All antibodies were specific for the methoxy subclass of mycolic acid with weak binding for α mycolic acid and did not show any binding to closely related lipids or other Mycobacterium tuberculosis (Mtb) derived lipids. We also determined the clinical utility of these antibodies based on their limit of detection for mycobacteria colony forming units (CFU). In combination with an optimized alkaline hydrolysis method for rapid lipid extraction, these antibodies can detect 10(5) CFU of Mycobacterium bovis BCG, a close relative of Mtb and therefore represent a novel approach for the development of diagnostic assays for lipid biomarkers.
authors.affiliation: Department of Microbiology, National University of Singapore, Singapore, 117545 Singapore.
authors.forename: Conrad E
authors.initials: CE
authors.lastname: Chan
authors.forename: Bryan Z
authors.initials: BZ
authors.lastname: Zhao
authors.forename: Amaury
authors.initials: A
authors.lastname: Cazenave-Gassiot
authors.forename: Shyue-Wei
authors.initials: SW
authors.lastname: Pang
authors.forename: Anne K
authors.initials: AK
authors.lastname: Bendt
authors.forename: Markus R
authors.initials: MR
authors.lastname: Wenk
authors.forename: Paul A
authors.initials: PA
authors.lastname: MacAry
authors.forename: Brendon J
authors.initials: BJ
authors.lastname: Hanson
mesh_terms: D000907:Antibodies, Bacterial / Q000096:biosynthesis / Q000737:chemistry*; D000918:Antibody Specificity; D015415:Biomarkers / Q000378:metabolism; D061505:Cell Surface Display Techniques; D004797:Enzyme-Linked Immunosorbent Assay; D057809:HEK293 Cells; D006801:Humans; D057230:Limit of Detection; D009163:Mycobacterium bovis / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000378:metabolism; D009171:Mycolic Acids / Q000276:immunology* / Q000302:isolation & purification / Q000378:metabolism; D011485:Protein Binding; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis* / Q000378:metabolism
chemical_list: D000907:Antibodies, Bacterial; D015415:Biomarkers; D009171:Mycolic Acids
keywords: antibody-based assay; biomarker; mycolic acid; phage display